K-ras protooncogene mutations in feline pancreatic adenocarcinomas.
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Adenocarcinoma↗
Biomedical subjects
G Schaffner
Publications and source records attributed to G Schaffner.
K-ras mutations in canine pancreatic cancers.
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Adenocarcinoma↗
N-ras mutations in canine malignant melanomas.
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Animals↗
N-ras mutation in a canine lymphoma: short communication.
Lymphomas of dogs were investigated by molecular genetic methods. Regions of exon 1 and 2 of the N-ras gene, which harbours the mutation hot spots (codons 12, 13 and 61) were screened. A GGT [symbol: see text] GAT (glycine [symbol: see text] aspartic acid) mutation in codon 13 was present in a multicentric-type lymphoma of a 1-year-old male dog.
Animals↗
N-ras mutation in a feline lymphoma. Low frequency of N-ras mutations in a series of feline, canine and bovine lymphomas.
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Animals↗
Tumour suppressor gene p53 mutation in a case of haemangiosarcoma of a dog.
Haemangiosarcomas of dogs were analysed by molecular genetic techniques. Regions of the tumour suppressor gene p53, including the well-known tumour hot spots (codons 175, 245, 248, 249, 273 and 282) were screened. A 24 bp deletion was detected in exon 5 of the gene.
Animals↗
A screen for genes required for meiosis and spore formation based on whole-genome expression.
BACKGROUND: Meiosis is the process by which gametes are generated with half the ploidy of somatic cells. This reduction is achieved by three major differences in chromosome behavior during meiosis as compared to mitosis: the production of chiasmata by recombination, the protection of centromere-proximal sister chromatid cohesion, and the monoorientation of sister kinetochores during meiosis I. Mistakes in any of these processes lead to chromosome missegregation. RESULTS: To identify genes involved in meiotic chromosome behavior in Saccharomyces cerevisiae, we deleted 301 open reading frames (ORFs) which are preferentially expressed in meiotic cells according to microarray gene expression data. To facilitate the detection of chromosome missegregation mutants, chromosome V of the parental strain was marked by GFP. Thirty-three ORFs were required for the formation of wild-type asci, eight of which were needed for proper chromosome segregation. One of these (MAM1) is essential for the monoorientation of sister kinetochores during meiosis I. Two genes (MND1 and MND2) are implicated in the recombination process and another two (SMA1 and SMA2) in prospore membrane formation. CONCLUSIONS: Reverse genetics using gene expression data is an effective method for identifying new genes involved in specific cellular processes.
Cell Nucleus↗
Genetic mapping with SNP markers in Drosophila.
Map-based positional cloning of Drosophila melanogaster genes is hampered by both the time-consuming, error-prone nature of traditional methods for genetic mapping and the difficulties in aligning the genetic and cytological maps with the genome sequence. The identification of sequence polymorphisms in the Drosophila genome will make it possible to map mutations directly to the genome sequence with high accuracy and resolution. Here we report the identification of 7,223 single-nucleotide polymorphisms (SNPs) and 1,392 insertions/deletions (InDels) in common laboratory strains of Drosophila. These sequence polymorphisms define a map of 787 autosomal marker loci with a resolution of 114 kb. We have established PCR product-length polymorphism (PLP) or restriction fragment-length polymorphism (RFLP) assays for 215 of these markers. We demonstrate the use of this map by delimiting two mutations to intervals of 169 kb and 307 kb, respectively. Using a local high-density SNP map, we also mapped a third mutation to a resolution of approximately 2 kb, sufficient to localize the mutation within a single gene. These methods should accelerate the rate of positional cloning in Drosophila.
Animals↗
Lack of sequence variation in sporadic bovine leucosis in regions of tumour suppressor genes p53 and p16.
Regions of the promoter and exons 5-8 of the tumour suppressor gene p53 were analysed in 25 cases of sporadic bovine leucosis. The study included 17 cases of juvenile leucosis, five cases of adult leucosis and three cases of skin leucosis. Exon 2 of tumour suppressor gene p16 was also investigated in the same samples. No sequence variations were present in the analysed areas of the genes. In p53, this fact represents a clear difference in comparison with enzootic bovine leucosis. In p16, no comparative data are available.
Amino Acid Sequence↗
Comparative studies in the promoter and exon 1 regions of tumour suppressor p53 in several mammalian species: absence of mutations in a panel of spontaneous domestic animal tumours.
Tumour suppressor p53 is critical in a broad panel of tumour types in human, mouse and other mammals. Regions of the promoter and exon 1 play an important role in expression of p53. In the present study, the DNA sequences of promoter and exon 1 regions of four domestic animal species (dog, cat, horse and cattle) are determined and compared with experimental rodents (mouse, rat and hamster) and man. A broad panel of tumour types have been investigated for mutations in this regulatory area in 90 canine, 136 feline, 25 equine and 10 bovine patients. No mutation was detected in any of the tumours analysed.
Animals↗
Presence of p53 mutations in feline neoplasms.
A region from exon 4 to 8 of the tumour suppressor gene p53 was analysed in 60 feline tumours (30 fibrosarcomas, seven malignant histiocytomas, three lymphosarcomas, five basal cell tumours, five squamous cell carcinomas, two adenocarcinomas of tubular skin glands, one undifferentiated carcinoma of the skin, seven mammary carcinomas). Missense mutations were detected in two fibrosarcomas, one malignant fibrous histiocytoma, the undifferentiated carcinoma of the skin and one mammary carcinoma. One nonsense mutation was detected in one fibrosarcoma and one deletion/frameshift-mutation was observed in one squamous cell carcinoma.
Animals↗
Novel p53 tumour suppressor mutations in cases of spindle cell sarcoma, pleomorphic sarcoma and fibrosarcoma in cats.
Twenty feline neoplasms were sequenced in the region from exons 5 to 8 for the presence of tumour suppressor gene p53 mutations. In a spindle cell sarcoma of the bladder, a missense mutation (codon 164 AAG-->GAG, lysine-->glutamic acid) in exon 5 was detected. In a pleomorphic sarcoma, a 23 bp deletion involving the splicing junction between intron 5 and exon 6 was observed. In a fibrosarcoma, a 6 bp deletion of p53 covering 2 bp of exon 7 and 4 bp of intron 7, including the splicing junction, was found. The study demonstrates three new p53 mutations in different types of sarcomas in cats.
Amino Acid Sequence↗
Podoplanin, novel 43-kd membrane protein of glomerular epithelial cells, is down-regulated in puromycin nephrosis.
Puromycin aminonucleoside nephrosis (PAN), a rat model of human minimal change nephropathy, is characterized by extensive flattening of glomerular epithelial cell (podocyte) foot processes and by severe proteinuria. For comparison of expression of glomerular membrane proteins of normal and PAN rats, a membrane protein fraction of isolated rat glomeruli was prepared and monoclonal antibodies were raised against it. An IgG-secreting clone designated LF3 was selected that specifically immunolabeled podocytes of normal but not of PAN rats. The antigen of LF3 IgG was identified as a 43-kd glycoprotein. Molecular cloning of its cDNA was performed in a delta gt11 expression library prepared from mRNA of isolated rat glomeruli. The predicted amino acid sequence indicated a 166-amino-acid integral membrane protein with a single membrane-spanning domain, two potential phosphorylation sites in its short cytoplasmic tail, and six potential O-glycosylation sites in the large ectodomain. High amino acid sequence identities were found to membrane glycoproteins of rat lung and bone and mouse thymus epithelial cells as well as to a phorbol-ester-induced protein in a mouse osteoblast cell line and to a canine influenza C virus receptor. In PAN, expression of this 43-kd protein was selectively reduced to < 30%, as determined by quantitative immunogold electron microscopy, immunoblotting, and Northern blotting. These data provide evidence that transcription of the 43-kd transmembrane podocyte glycoprotein is specifically down-regulated in PAN. To indicate that this protein could be associated with transformation of arborized foot processes to flat feet (Latin, pes planus) we have called it podoplanin.
Amino Acid Sequence↗
The complete DNA sequence and genomic organization of the avian adenovirus CELO.
The complete DNA sequence of the avian adenovirus chicken embryo lethal orphan (CELO) virus (FAV-1) is reported here. The genome was found to be 43,804 bp in length, approximately 8 kb longer than those of the human subgenus C adenoviruses (Ad2 and Ad5). This length is supported by pulsed-field gel electrophoresis analysis of genomes isolated from several related FAV-1 isolates (Indiana C and OTE). The genes for major viral structural proteins (Illa, penton base, hexon, pVI, and pVIII), as well as the 52,000-molecular-weight (52K) and 100K proteins and the early-region 2 genes and IVa2, are present in the expected locations in the genome. CELO virus encodes two fiber proteins and a different set of the DNA-packaging core proteins, which may be important in condensing the longer CELO virus genome. No pV or pIX genes are present. Most surprisingly, CELO virus possesses no identifiable E1, E3, and E4 regions. There is 5 kb at the left end of the CELO virus genome and 15 kb at the right end with no homology to Ad2. The sequences are rich in open reading frames, and it is likely that these encode functions that replace the missing El, E3, and E4 functions.
Adenoviruses, Human↗
Cancer vaccines: the interleukin 2 dosage effect.
Cancer vaccines genetically engineered to produce interleukin 2 have been investigated intensively in a series of animal models and are at the point of entering into clinical trials. In this study we demonstrate a strong correlation between the rate of interleukin 2 production and the protection efficiency of murine S91 melanoma cell (clone M-3) vaccines. Best immunization is achieved with vaccines producing medium interleukin 2 levels of 1000-3000 units per 10(5) cells per day. Reduced interleukin 2 production evokes a corresponding decline in the number of successfully treated animals. Unexpectedly, when interleukin 2 expression is raised to high levels of 5000-7500 units per 10(5) cells per day, protection is completely absent because of impaired generation of tumor-specific cytotoxic T lymphocytes. In comparison, granulocyte-macrophage colony-stimulating factor as immunomodulator induces substantial immunization even at a moderate level of secretion and protects all animals at the maximal obtainable level of secretion. Our findings demonstrate the importance of the interleukin 2 level produced by genetically modified tumor cells and may have substantial impact for the clinical application of cancer vaccines.
Animals↗
A novel class of autoantigens of anti-neutrophil cytoplasmic antibodies in necrotizing and crescentic glomerulonephritis: the lysosomal membrane glycoprotein h-lamp-2 in neutrophil granulocytes and a related membrane protein in glomerular endothelial cells.
Necrotizing and crescentic glomerulonephritis (NCGN) is frequently associated with circulating antineutrophil cytoplasmic autoantibodies (ANCA). It is established that ANCA are specific for soluble enzymes of granules of polymorphonuclear neutrophil granulocytes (PMN), such as myeloperoxidase (MPO) or protease 3 (PR3). The purpose of this study was to identify membrane proteins of PMNs, and/or glomerular cells, as additional autoantigenic ANCA targets. When membrane protein fractions were prepared from PMNs and isolated human glomeruli, and immunoblotted with ANCA sera of NCGN patients, two bands with apparent molecular masses of 170 and 80-110 kD (gp170/80-110) were labeled in PMNs, and a 130-kD glycoprotein (gp130) in glomeruli. Gp130 was purified, and monoclonal and rabbit antibodies (Abs) were produced which showed the same double specificity as the patient's ANCA. Using these probes, evidence was provided that gp170/80-110 is identical with human lysosomal-associated membrane protein 2 (h-lamp-2), because both proteins were immunologically cross-reactive and screening of a cDNA expression library from human promyelocytic leukemia cells with anti-gp130 Ab yielded a clone derived from h-lamp-2. Gp170/80-110 was localized primarily in granule membranes of resting PMNs, and was translocated to the cell surfaces by activation with FMLP. By contrast, gp130 was localized in the surface membranes of endothelial cells of human glomerular and renal interstitial capillaries, rather than in lysosomes, as found for h-lamp-2. Potential clinical relevance of autoantibodies to gp170/80-110 and gp130 was assessed in a preliminary trial, in which ANCA sera of patients (n = 16) with NCGN were probed with purified or recombinant antigens. Specific reactivity was detected in approximately 90% of cases with active phases of NCGN, and frequently also in combination with autoantibodies specific for PR3 or MPO. Collectively, these data provide evidence that h-lamp-2 in PMNs and a different, structurally related 130-kD membrane protein on the cell surface of renal microvascular endothelial cells are autoantigenic targets for ANCA in patients with active NCGN.
Amino Acid Sequence↗
Receptor-mediated gene transfer into human T lymphocytes via binding of DNA/CD3 antibody particles to the CD3 T cell receptor complex.
Retrovirus-mediated gene transfer is currently the method of choice for the transfection of human T lymphocytes for applications in gene therapy. Use of retroviral vectors, however, is hampered by limits on the size of the genetic material to be transferred, the requirement of dividing target cells, and by potential safety questions. Synthetic peptide-enhanced or adenovirus-enhanced receptor-mediated transferrinfection of DNA (SPET and AVET, respectively) is a powerful method for the introduction of genetic material into mammalian cells. Although transferrin has proven to be a useful ligand for gene transfer in many cell types, gene expression in T cells with transferrin/DNA complexes is usually not satisfactory. To improve gene transfer to T cells, antibodies directed against the CD3-T cell receptor complex were tested for their ability to function as ligands for DNA delivery. In T cell lines, up to 50% of the cells expressed a beta-galactosidase reporter gene using anti-CD3 gene transfer complexes. Applying optimized conditions, prestimulated primary peripheral blood lymphocytes were also transfected successfully, although at a lesser efficiency (5%).
Antibodies↗
DNA sequence recognition by Pax proteins: bipartite structure of the paired domain and its binding site.
Previous DNA-binding studies indicated that an intact paired domain is required for interaction of the transcription factor BSAP (Pax-5) with DNA. We have now identified a subset of BSAP recognition sequences that also bind to a truncated BSAP peptide lacking 36 carboxy-terminal amino acids of the paired domain. Sequence comparison of this class of BSAP-binding sites made it possible to unequivocally align all known BSAP-binding sites and to deduce a consensus sequence consisting of two distinct half sites. We propose here a model for the paired domain--DNA interaction in which the paired domain is composed of two subdomains that bind to the two half-sites in adjacent major grooves on the same side of the DNA helix. The existence of these half sites and of the two paired domain subregions was directly demonstrated by methylation interference analysis and by in vitro mutagenesis of both the paired domain and its recognition sequence. Both half-sites contribute to the overall affinity of a given BSAP-binding site according to their match with the consensus sequence. However, none of the naturally occurring BSAP-binding sites completely conform to the consensus sequence. Instead, they contain compensatory base changes in their half-sites that explain the versatile and seemingly degenerate DNA sequence recognition of Pax proteins. Domain swap experiments between BSAP and Pax-1 demonstrated that the sequence specificity of the BSAP paired domain is determined by both its amino- and carboxy-terminal subdomains. Moreover, mutations affecting only one of the two subdomains restricted the sequence specificity of the paired domain. Such mutations have been shown previously to be the cause of mouse developmental mutants (undulated, Splotch, and Small eye) and human syndromes (Waardenburg's syndrome and aniridia) and may thus differentially affect the regulation of target genes by the mutated Pax protein.
Base Sequence↗